1. DOPAMINERGIC AMACRINE CELLS
EXPRESS OPIOID RECEPTORS IN THE
MOUSE RETINA
Shannon K. Gallagher,1 Julia N. Anglen,1 Justin M. Mower,1 and Jozsef Vigh1*
1Department of Biomedical Sciences, Colorado State University, Fort Collins, CO
80523, USA.
*Correspondence: Jozsef.Vigh@Colostate.edu
doi:10.1017/S0952523812000156.
2. ABSTRACT
• Opioid receptors confirmed to exist by a variety of techniques in vertebrae
mammals
• However location only found in retinal regions that specific cell types
• Current knowledge of opioid signaling limited by only few studies
• Best documented opioid effect is modulation of retinal dopamine release
• Not known if opioids can affect dopamanergic amacrine cells (DACS) via
opioid receptors expressed by DACS
• They found through immunohistochemical methods, determine whether
• μ- and δ-opioid receptors (called MORs and DORs, respectively) are present in the
mouse retina
• Are these expressed by the DACS
• Found MOR & DOR immunolabeling was associated with many cell-types in
the inner retina
• Opioids suggest influence visual information processing at multiple sites in
mammalian retinal circuitry
• DAC molecular marker labeling shows anti-tyrosine hydroxylase antibody shows
MOR & DOR immunolabeling localize to DAC
• Predict opioids cann affect DACS in mouse retina directly by MOR & DOR
signaling (and might modulate dopamine release as reported in other
mammals and nonmammals
3. INTRODUCTION
• Mammalian retina opioids
• regulate the cell proliferation during development
• Influence cell survival after hypoxic or ischemia
• Regulate dopamine release via MOR and DOR activation
• Endogenous opioids play an important role in processing
sensory information
• Only sporadic data suggest these exist in mammalian retina
• Enkephalin detected in inner retinal neurons of guinea pigs & rat
retinal extract
• Three classes of opioid receptors do not show exclusive endogenous
substrate specificity
• β-endorphin binds preferentially to μ-opioid receptors (MORs)
• enkephalins to δ-opioid receptors (DORs)
• dynorphins to κ-opioid receptors (KORs)
• Peng et al (2009)
• presence of both MORs and DORs through RT-PCR and Western blot analysis,
• Brecha et al. (1995)
• MORs were also detected by immunohistochemistry on processes of bistratified
ganglion cells
5. ANIMALS
• Mice Used
• Adult male and female, wild type
• Kept in 12 hour light, 12 hour dark cycle
• Sprague-Dawley Dams
• Immunohistochemical procedures
• Using antibodies to observe
6. IMMUNOHISTOCHEMISTRY
• Refers to detecting antigens in a specific tissue
cell section by allowing the antibodies to
specifically bind to antigens in biological tissues
• Done to help observe distribution and localiaztion of
biomarkers and different proteins
• On sections
• Retina-, Brain-, Dorsal root ganglia (DRG)
7. ANTIBODIES
• Antibodies
• (also known as immunoglobulin)
• is a protein used by the immune system to identify and neutralize foreign
objects like bacteria and viruses
• The antibody recognizes a specific target called the antigen
• Antibodies used
• Raised against Brn-3a
• synthetic peptide corresponding to the N-terminus region of human Brn-
3a
• Raised against δ-opioid receptors (DORs)
• synthetic peptide corresponding to amino acids
• N-terminus of mouse DOR
• Raised against μ-opioid receptors (MORs)
• peptide corresponding to amino acids
• synthetic peptide corresponding to amino acids
• Raised against Tyrosine Hydroxylase (TH)
• mouse anti-TH monoclonal antibody
8. CONFOCAL LASER MICROSCOPY
• Technique for getting high-resolution optical imates with
depth selectivity
• Key feature is to see in-focuse images from selected depths called
optical sectioning
9. Figure 2.
Some ganglion cells and GABAergic amacrine cells are MOR+. A: 40x single-plane merged image of vertically sectioned GAD67-
EGFP mouse retina immunolabeled for MOR (red; Alomone). GAD67-EGFP somas are seen in the INL (bright green) and GCL (dim
green) with processes in the IPL. Punctate MOR+ labeling of a displaced GABAergic amacrine cell is shown in the GCL (arrow). Some
putative MOR+ somas in the GCL are GAD67-EGFP negative (arrowheads). In the INL some GAD67-EGFP cells colabel MOR+
puncta that could indicate colocalization (asterisks). B: 40x merged confocal image of cryosectioned wild-type mouse retina co-
immunolabeled for MOR (red) and Brn-3a (green). Some Brn-3a + retinal ganglion cells (arrow), but not all (arrowhead), are MOR+.
INL: inner nuclear layer; IPL: inner plexiformlayer; GCL: ganglioncell layer. Scale bars:20μm.
11. μ-OPIOID RECEPTORS IN MOUSE
RETINA
• Might be some types of MOR bearing cells due to diverse
size & morphology of MOR+ somas
• MOR immunolabeling sometimes colocalized with the
GAD67-EGFP
12. δ-OPIOID RECEPTORS IN MOUSE
RETINA
• DADLE
• a synthetic enkephalin
• reduces dopamine release is considered to be a δ- opioid
receptor (DOR)-selective agonist
• Colabeling studies with anti-TH antibody indicated that
one of the DOR+ retinal cell types is the DAC
13. TH+ dopaminergic processes
• Showed neither MOR nor DOR immounolabeling
• No colocalization detected within the IPL
14. Figure 1
• Figure 1.
Immunohistochemical localization of MORs
in mouse retinal and hippocampal tissues.
A: 40x confocal single-plane image of
vertical cryosectioned mouse retina showing
immunolabeling with anti-MOR antibody
directed against the N-terminus of MOR
(Alomone). MOR+ puncta are observed in
the inner retina with discernible cells labeled
in the INL and GCL (arrows). B: 40x image
similar to A showing control peptide
preadsorption for MOR antibody. C: 10x
confocal image of mouse brain slice
focusing on the hippocampus. MOR
antibody showing appropriate
immunolabeling (green), colabeled with the
nuclear marker ToPro3 (red). D: 40x focused
confocal image of the CA3 region of mouse
hippocampus immunolabeled for MORs
(green), colabeled with ToPro3 (red). E: 40x
image similar D showing preadsorption of
MOR antibody with control
peptide, colabeled with ToPro3 (red). ONL:
outer nuclear layer; OPL: outer plexiform
layer; INL: inner nuclear layer; IPL: inner
plexiform layer; GCL: ganglion cell layer;
DG: dentate gyrus. Scale bars: A, B, D, and
15. Figure 3
• Figure 3.
Dopaminergic amacrines in the INL are
MOR+. A: 40x single-plane image of
vertically sectioned mouse retina showing
MOR+ (red) puncta in the INL (arrow) using
the N- terminus directed MOR antibody
(Alomone). B: Image displaying the same
retinal region as A, immunolabeled for TH
(green) showing a single TH+ cell (arrow) in
the INL with TH+ projections in the IPL at the
border with the INL. C: A merged image of A
and B, displaying colocalization of the
MOR+ and TH+ cell (arrow). D: 40x confocal
image, vertical section of mouse retina
showing immunolabeled somata (red) in the
INL (arrow) with the anti-MOR antibody
directed against the C-terminus of the
receptor (Epitomics). E: Image illustrating
the same region as in D, showing a TH+
(green) soma in the INL (arrow). F: A
merged image of D and E, indicating
colocalization of MOR and TH
immunolabeling. INL: inner nuclear layer;
IPL: inner plexiform layer; GCL: ganglion cell
layer. Dashed lines (C) demarcate example
focused images used for colocalization
analysis (see Methods). Scale bars: 20μm.
16. Figure 4
• Figure 4.
Localization of DOR immunolabeling
in mouse retinal and rat dorsal root
ganglion tissues. A: 40x confocal
image of cryosectioned mouse retina
immunolabeled with an anti-DOR
antibody (Alomone). Note the puncta
in the inner retina with putative
somatic labeling in the INL (arrows).
B: 40x image similar to A showing
control peptide preadsorption for DOR
(Alomone) antibody. C: 40x confocal
image of rat DRG with DOR+ somas
(green). Colabled with the nuclear
marker ToPro3 (red). D: 40x image
similar C showing preadsorption of
DOR antibody with control
peptide, colabeled with ToPro3 (red).
ONL: outer nuclear layer; OPL: outer
plexiform layer; INL: inner nuclear
layer; IPL: inner plexiform layer; GCL:
ganglion cell layer. Scale bars: 20μm.
17. Figure 5
• Figure 5.
Multiple inner retinal cell-types including
dopaminergic amacrines are DOR+. A: 40x
single-plane confocal image, vertical section of
mouse retina showing DOR+ (red; Alomone)
somata (arrow). B: Image displaying the same
region as A, immunolabeled for TH (green). C: A
merged image of A and B, showing a DOR+ and
TH+ amacrine cell in the INL (arrow). D: 40x
single-plane merged confocal image of vertically
sectioned wild-type mouse retina
coimmunolabeled for DOR (red; Millipore) and
Brn-3a (green). Some Brn-3a+ retinal ganglion
cells are MOR+ (arrow). Arrowhead indicating a
putative DOR+ soma in the INL. E: A 40x single-
plane merged confocal image of cryosectioned
GAD67-EGFP mouse retina co-immunolabeled
for DOR (red; Millipore) and TH (blue). GAD67-
EGFP somas are seen in the INL (bright green)
and GCL (dim green) with processes in the IPL. A
GABAergic (EGFP+) displaced amacrine cell in
the GCL is DOR+ (arrow). Some putative DOR+
somas in the GCL are GAD67-EGFP negative
(arrowhead). In the INL, a TH+ soma (blue) is
DOR+ (asterisks). F: Focused view of
dopaminergic amacrine cell from E showing that
the TH+ soma is EGFP- and DOR+. INL: inner
nuclear layer; IPL: inner plexiform layer; GCL:
ganglion cell layer. Scale bars: C, D, and E:
20μm, F: 10μm.
18. DISCUSSION
• MOR immunolabeling in mouse retina
• Subpopulation of Brn-3a+ GCs (Fig. 2B), GAD67+ GABAergic ACs (Fig.
2A), and dopaminergic amacrine cells (DACs) express MORs (Fig. 3)
• DOR immunolabeling in mouse retina
• Subpopulation of Brn-3a+ GCs (Fig. 5D) and GAD67-expressing
GABAergic amacrines (Fig. 5E) express DORs+ inner retinal cell typs
(Fig. 4A)
• Data confirms that DACs are DOR+
• The majority of opioid receptor activity is mediated
through the Go/Gi -coupled superfamily of receptors, and
the cellular effects include:
• activation of inwardly rectifying potassium current
• inhibition of voltage-gated calcium current
• inhibition of adenylate cyclase (depending on the cell type)
• TH+ dopaminergic processes did not have MOR nor DOR
immounolabeling
• Not unprecedented
19. GALLAGHER, S. K., ANGLEN, J. N., MOWER, J. M., & VIGH, J.
(2012). DOPAMINERGIC AMACRINE CELLS EXPRESSK
OPIOID RECEPTORS IN THE MOUSE RETINA. VIS
NEUROSCI, 203-209. RETRIEVED FROM HTTP://
WWW.NCBI.NLM.NIH.GOV/PMC/ARTICLES/PMC3367769/
